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- PDB-6ee8: Mycobacterium tuberculosis RNAP promoter unwinding intermediate c... -
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Basic information
Entry | Database: PDB / ID: 6ee8 | ||||||
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Title | Mycobacterium tuberculosis RNAP promoter unwinding intermediate complex with RbpA/CarD and AP3 promoter | ||||||
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![]() | TRANSCRIPTION/DNA / initiation / half bubble / open promoter complex / TRANSCRIPTION / intermediate / TRANSCRIPTION-DNA complex | ||||||
Function / homology | ![]() bacterial-type RNA polymerase holo enzyme binding / response to water / Antimicrobial action and antimicrobial resistance in Mtb / bacterial-type RNA polymerase core enzyme binding / sigma factor activity / rRNA transcription / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / ribonucleoside binding ...bacterial-type RNA polymerase holo enzyme binding / response to water / Antimicrobial action and antimicrobial resistance in Mtb / bacterial-type RNA polymerase core enzyme binding / sigma factor activity / rRNA transcription / DNA-directed RNA polymerase complex / peptidoglycan-based cell wall / DNA-templated transcription initiation / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / nucleic acid binding / protein dimerization activity / response to antibiotic / DNA-templated transcription / positive regulation of DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / plasma membrane / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.92 Å | ||||||
![]() | Darst, S.A. / Campbell, E.A. / Boyaci Selcuk, H. / Chen, J. | ||||||
![]() | ![]() Title: Structures of an RNA polymerase promoter melting intermediate elucidate DNA unwinding. Authors: Hande Boyaci / James Chen / Rolf Jansen / Seth A Darst / Elizabeth A Campbell / ![]() ![]() Abstract: A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex. To generate the open complex, the conserved catalytic core of the RNAP combines ...A key regulated step of transcription is promoter melting by RNA polymerase (RNAP) to form the open promoter complex. To generate the open complex, the conserved catalytic core of the RNAP combines with initiation factors to locate promoter DNA, unwind 12-14 base pairs of the DNA duplex and load the template-strand DNA into the RNAP active site. Formation of the open complex is a multi-step process during which transient intermediates of unknown structure are formed. Here we present cryo-electron microscopy structures of bacterial RNAP-promoter DNA complexes, including structures of partially melted intermediates. The structures show that late steps of promoter melting occur within the RNAP cleft, delineate key roles for fork-loop 2 and switch 2-universal structural features of RNAP-in restricting access of DNA to the RNAP active site, and explain why clamp opening is required to allow entry of single-stranded template DNA into the active site. The key roles of fork-loop 2 and switch 2 suggest a common mechanism for late steps in promoter DNA opening to enable gene expression across all domains of life. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 689.2 KB | Display | ![]() |
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PDB format | ![]() | 540.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 912.3 KB | Display | ![]() |
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Full document | ![]() | 953 KB | Display | |
Data in XML | ![]() | 91.4 KB | Display | |
Data in CIF | ![]() | 142.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 9039MC ![]() 9037C ![]() 9041C ![]() 9047C ![]() 6edtC ![]() 6eecC ![]() 6m7jC C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-DNA-directed RNA polymerase subunit ... , 4 types, 5 molecules ABCDE
#1: Protein | Mass: 37745.328 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() References: UniProt: A5U8D3, UniProt: P9WGZ1*PLUS, DNA-directed RNA polymerase #2: Protein | | Mass: 125390.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() References: UniProt: V9Z879, UniProt: P9WGY9*PLUS, DNA-directed RNA polymerase #3: Protein | | Mass: 148202.219 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() References: UniProt: A5U053, UniProt: P9WGY7*PLUS, DNA-directed RNA polymerase #4: Protein | | Mass: 11776.996 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Gene: rpoZ, ERS007672_03979, ERS007703_04032, ERS007720_04749, ERS027652_00548, ERS027654_02543, ERS027656_03959, ERS124361_02246 Production host: ![]() ![]() References: UniProt: A0A0T9N9K3, UniProt: P9WGY5*PLUS, DNA-directed RNA polymerase |
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-Protein , 1 types, 1 molecules F
#5: Protein | Mass: 58169.477 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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-RNA polymerase-binding ... , 2 types, 2 molecules JM
#6: Protein | Mass: 12993.695 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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#9: Protein | Mass: 17933.361 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
-DNA chain , 2 types, 2 molecules OP
#7: DNA chain | Mass: 27907.809 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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#8: DNA chain | Mass: 27618.625 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
-Non-polymers , 2 types, 3 molecules ![](data/chem/img/ZN.gif)
![](data/chem/img/MG.gif)
![](data/chem/img/MG.gif)
#10: Chemical | #11: Chemical | ChemComp-MG / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Mycobacterium tuberculosis RNAP promoter unwinding intermediate Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT |
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Source (natural) | Organism: ![]() ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 8 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Details: unspecified |
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 1.4 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.13_2998: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 140333 / Symmetry type: POINT | ||||||||||||||||||||||||
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